As in all experimental biomedical research the numbers of individuals (persons or animals) or samples, termed the experimental units, examined in this thesis are relatively small. Still, proper statistical handling of the data is crucial to draw scientific conclusions. The applied statistical methods are described in the individual papers, but a few key issues regarding the choice of the methods are discussed here.
As the experimental units are relatively few and consequently the sample sizes of each experimental group are small, a decision has to be made whether to apply parametric
statistical tests or non-parametric tests. If all assumptions for parametric tests are met, parametric tests offer greater ability to detect a true significant difference between groups (Lamb et al., 2008). The main assumption for using parametric tests is a normal distribution of data within each sample group. There is no clear definition of when to regard a data set normally distributed. Formal statistical tests for normality exist, but these are considered unnecessary (Lamb et al., 2008). Based on literature and consultations with biostatisticians we considered data set normally distributed if the visual impression of symmetry around the mean in columnar scatter plots was evident; and if mean and median values were
approximately equal; and if the nature of data indicated normality. No minimal number of experimental units per experimental group was set as a requirement for assuming normality.
Other assumptions on which the applied statistical tests are based are: that the experimental
units are either randomly selected from a population or randomly allocated to the experimental group; that the experimental units are independent of each other both within groups and across the groups; and that the variances between groups are relatively equal (Lamb et al., 2008). Except for the procedure of the stabling of mice which could be questioned with regard to independency (see section 5.2.5), these assumptions were met for all our analyses where parametric statistics where applied.
A disparity exists between papers Iand IIin terms of applied statistical method considering that assessments performed on partially the same specimens. When designing the project resulting in papers I and II we regarded the sample sizes too small for parametric tests and we therefore initially performed non-parametric tests. However, when analyzing data for paper IIwe considered the possibility of adjusting the cell density counts for inflammation score. We discussed this with a statistician (co-author J.M.G), and the data were judged to be eligible for parametric statistics.
Comparisons between two independent groups were analyzed with independent (a.k.a unpaired) tests (e.g. Student t test, Mann-Whitney). When comparing more than two groups these were analyzed with analysis of variances (ANOVA) rather than multiple t tests. The ANOVA tests reduce the chances of statistically significant results to erroneously occur by chance (type I error). Relevant post-tests were performed with Bonferroni adjustment.
ANOVA tests require assumptions of normality to be met.
In paper Iand IIwe performed multiple tests and not ANOVA. The choice of test was discussed with a statistician and considered valid because a number of null hypotheses were defined and decided to be tested prior to the acquisition of data, and because not all comparisons between various groups were considered relevant (e.g. adult colon vs. pediatric ileum).
All tests were performed as two-tailed tests and the level of significance (ĮOHYHOZDV consistently set to the conventional 0.05.
6 DISCUSSION
Discussions of the isolated results of the individual papers are provided in the respective manuscripts. This chapter provides an integrated discussion of the results in relation to the aims of the thesis.
This thesis aimed at providing new information on mucosal immunopathogenic mechanisms in IBD. Papers IandIIpresent novel observations on the distribution of macrophages and Tregs cells in the mucosa of recent onset pediatric IBD patients compared with recent onset adult IBD patients. The study design founding the basis for these papers is original, as at the time of diagnosis biological material from untreated patients were collected.
This provided insight into the early pathogenesis of IBD. Most investigations of biologic material from IBD patients have been based on collections from patients with long-standing disease and concurrently varying medication. To our knowledge the two papers included here are the first to have investigated mucosal specimens from exclusively untreated pediatric patients at the time of diagnosis and having compared them with specimens from untreated adult patients at the time of diagnosis.
Papers Iand IIreport on differential distribution of macrophages and Tregs, both important players in intestinal mucosal homeostasis (Hooper and Macpherson, 2010). We suggest that these differences may contribute to phenotypic differences in pediatric and adult onset IBD. This hypothesis is based on the assumption that there is no qualitative difference in the function of these cell types in pediatric versus adult patients. Regarding macrophages, researchers have proposed that CD is the result of impaired macrophage function (Casanova and Abel, 2009). Dysfunction of Tregs has not been linked to development of IBD, but the increasing knowledge of plasticity between Tregs and Th17 effector cells makes it difficult to assess the exact role of Foxp3+Tregs in the etiology and pathogenesis of IBD (Sakaguchi et al., 2010).
Papers Iand IIdescribe that the pediatric control patients have fewer macrophages in their colon and that the pediatric CD patients have more Tregs in their ileum compared with adults, but no mechanistic explanations are provided. As we have demonstrated in the included Papers IV andVand consistent with the current literature, nearly all experimental IBD animal models depend on the presence of intestinal microbes (Uhlig and Powrie, 2009).
Furthermore, both experimental and human IBD are associated with alterations in the
composition of the intestinal microbiota (Sartor, 2008). Is it possible that the altered
distribution of immune cells described in Papers Iand IIhas been caused by or is associated with microbial perturbations? Few investigations have been performed on the microbiota in pediatric IBD and none have identified alterations specific to the pediatric segment (Conte et al., 2006; Schwiertz et al., 2010). However, as recent animal studies have identified key phylotypes responsible for the induction of Tregs (see section 2.3.5), the hypothesis that the differential distribution of macrophages and Tregs is instigated by pediatric-specific microbes, which ultimately may cause the pediatric-specific IBD phenotypes, deserves to be explored.
The methodology of papers IandIIdid not allow functional assessments of the role of macrophages and Tregs in the IBD lesions. It is well documented that activated (CD40+) macrophages contribute to drive inflammation (Mosser and Edwards, 2008; Smith et al., 2010). It is therefore likely that the increased density of activated macrophages leads to increased inflammatory potential in the IBD lesion. Regarding Tregs, it may seem counterintuitive that there are many studies along with ours that report that Tregs are generally increased in numbers in all kinds of inflammatory lesions. Our analysis of the distribution of Tregs in the intestinal mucosa indicated that there are spatial differences in the distribution of Tregs regardless of the inflammatory status of the examined biopsy.
Experimental studies on the inflammatory potential in lesions with or without Tregs are not possible to perform in humans. However, humans with mutations in FOXP3(IPEX) have IBD-like enteropathy, and experimental animal studies have demonstrated that Tregs are able to both prevent colitis and attenuate established disease (Fantini et al., 2006; Mottet et al., 2003). Thus, the functional role of the cell types studied in papers Iand IIshould not be controversial. The applied immunohistochemistry methods provide a snapshot of real life distribution of key players in mucosal homeostasis.
Papers III-IVfocus on the roles of colonic epithelial cells and the transport of SIg in laboratory animal models. These are essential components of mucosal homeostasis along with macrophages and Tregs. The homeostatic function of IEC, IgA, macrophages, and Tregs all pivot around a few key cytokines such as TGF-ȕ, IL-10, TSLP and RA. Recently,
experimental studies have presented evidence for Tregs’ promotion of IgA production, a.k.a as a Treg-IgA axis (Feng and Elson, 2011).
The second aim of this thesis was to elucidate interactions between secretory immunoglobulins and intestinal epithelial cell function on one hand and the intestinal
microbiota on the other.Paper IVshows how lack of SIg makes mice more susceptible to experimental colitis and that this is associated with alterations of the composition of the intestinal microbiota and of the colonic IEC gene expression profile. These results are consistent with previously published data in mice (Murthy et al., 2006). However, as
previously mentioned, data recorded in mice cannot automatically be extrapolated to humans.
There are well known differences in the IgA system between mice and men (reviewed by Gibbons and Spencer (Gibbons and Spencer, 2011). In humans, no mutation resulting in aborted function of pIgR is known. From an evolutionary point of view, this may indicate that lack of pIgR is incompatible with life in humans as opposed to in mice. Alternatively, a defective pIgR may have little consequence in healthy humans living in a clean environment and therefore not have been identified. There are known single nucleotide polymorphisms in the human PIGRgene (2007). Some of these correlate with IgA nephropathy and with Epstein-Barr virus diseases, but none have been shown to correlate with IBD or other intestinal diseases.
On the other hand, selective IgA deficiency is well described in humans with a prevalence of 1:200 to 1:1000 in western societies. IgA deficiency leaves most of the affected persons asymptomatic, but it is still associated with increased incidence of IBD (Cunningham-Rundles, 2001). The reason for the mild disease phenotype in selective IgA deficiency is not established, but one hypothesis suggest that increased levels of SIgM could compensate for the lack of SIgA (Cunningham-Rundles, 2001). However, also patients with X-linked agammaglobulinemia, which lack all classes of immunoglobulins, rarely present with gastrointestinal disorders (Agarwal and Mayer, 2009). This suggests that isolated loss of immunoglobulins in humans is not critical to gut homeostasis, and consequently that there is redundancy to humoral immunity for maintenance of mucosal homeostasis. In humoral immunodeficencies such as common variable immunodeficiency, it is believed that intestinal inflammation is dependent on concurrent impairment of T cell function (Agarwal and Mayer, 2009). Also, mice gut homeostasis is dependent on T cell function (Gibbons and Spencer, 2011). One study has demonstrated the importance of Tregs for mediating OVA specific tolerance in pIgR KO (Karlsson et al., 2010), but further reports on T cell functions in the pIgR KO and JHKO mouse models are lacking.
Paper IVoutlines changes in the composition of the intestinal microbiota in mice deficient in SIgs. As previously mentioned, several new pieces of evidence indicate how the intestinal microbiota is shaped by SIgA. Reports on how humoral immunodeficiency affects
the intestinal microbiota in humans are sparse, and to our knowledge the microbiota of these patients have not been assessed with modern phylogenetic tools. Such studies would be of great interest as they could validate the relevance of the observations in mice and help with identifying defined phylotypes that exploit the lack of SIgs to establish their niche.
To what extent do the results from this thesis provide information that can be implemented into efforts to prevent IBD or improve the care of affected patients?
Immunosuppressive therapy by enhancing Treg function based on principles of ex vivo expansion and reinfusion is under intense research (Sakaguchi et al., 2010). The finding of increased density of Tregs in an intestinal segment of pediatric intestines with reduced occurrence of CD lesions (paper II) should encourage continuation of this line of research.
Our results in the mouse models (papers III-V) confirm previous reports that colitis is dependent on presence of intestinal microbes, and we showed that mice with increased susceptibility to colitis had an altered composition of the microbiota. However, since these mice were lacking SIgs and pIgR-mediated mucosal protection, we cannot determine whether enhanced susceptibility to DSS-induced colitis was due to altered microbiota or altered handling of the present microbes. Key phylotypes that potentially cause IBD have yet to be identified. The idea of modulating the intestinal microbiota to treat IBD has been pursued.
Probiotics have shown promising results in ulcerative colitis, but larger clinical trials are still lacking. Recent reports on specific phylotypes responsible for Treg induction in the gut of mice may give a clue to key species that a probiotic concoction should contain (Atarashi et al., 2011). Allogenic fecal transplantation, which is becoming established therapy for resistant C. difficileinfections, has been tested in IBD patients with good results (Khoruts and Sadowsky, 2011). Recent data in an experimental mouse model demonstrated that colitogenic bacterial strains can be transferred vertically (mother to pup) and horizontally (between litter mates) in mice (Garrett et al., 2007; Garrett et al., 2010). Fecal transplantation from a healthy to an affected individual is the therapeutic analogue to those observations and should be explored more thoroughly despite esthetical concerns. Our results along with other experimental data on the interaction between IgA and intestinal microbiota (Peterson et al., 2007; Suzuki et al., 2004) suggest that IBD patients with impaired humoral immunity should in particular benefit from restoration of a healthy gut microbiota.
7 CONCLUSIONS
Intensive research over the past few decades has demonstrated that IBD is associated with disrupted homeostasis in the intestinal mucosa with alterations in both host immunity and in the intestinal microbiota. This thesis provides the following new information to the ever increasing knowledge on disrupted intestinal mucosal homeostasis:
x Histologically normal colonic mucosa has a higher density of macrophages in children than in adults.
x Activated mucosal macrophages are increased in untreated pediatric IBD.
x The majority of CD25+cells in intestinal mucosa are macrophages.
x Densities of mucosal Foxp3+Tregs and CD25+cells (activated macrophages) are elevated in both pediatric and adult ileal CD regardless of histological inflammation.
x Cultivatable intestinal microbiota can be depleted by gavaging antibiotics, and the biological effect of this treatment phenocopies physiological characteristics of germ-free mice.
x Mice lacking SIg because of abrogated transport of pIg have altered composition of the intestinal microbiota, and this is associated with increased susceptibility to experimental colitis and differential gene expression profile of colonic IECs.
x Absence of pIgR protects B cell-deficient mice from microbiota-dependent colitis.
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